David J. Uhlinger

RhoA and a Cytosolic 50-kDa Factor Reconstitute GTPγS-dependent Phospholipase D Activity in Human Neutrophil Subcellular Fractions

Receptor activation of phospholipase D has been implicated in signal transduction in a variety of cells. Reconstitution of cell-free guanosine 5′-O-(3-thiotriphosphate)(GTPγS)-dependent phospholipase D activity from human neutrophils requires protein factors in both the plasma membrane and the cytosol. We previously proposed that one of the factors is a Ras-family small molecular weight GTPase of the Rho subtype (Bowman, E. P., Uhlinger, D. J., and Lambeth, J. D.(1993) J. Biol. Chem. 268, 21509-21512). Herein, we have used RhoGDI (GDP dissociation inhibitor), an inhibitory Rho-binding protein, to selectively extract Rho-type GTPases from the plasma membrane, and have used immunoprecipitation as well as chromatographic methods to remove cytosolic Rho. Depletion of RhoA from either the plasma membrane or the cytosol resulted in a partial loss in GTPγS dependent activity, while removal of RhoA from both fractions resulted in a nearly complete loss in activity. Activity was nearly completely restored by adding purified recombinant RhoA, which showed an EC50 of 52 nM, while Rac1 showed little activity. Cytosol fractionated using DEAE-cellulose chromatography separated ADP-ribosylation factor and Rho from the major activating fraction. Gel exclusion chromatography of this fraction revealed an activating factor of 50 kDa apparent molecular mass. Using RhoA-depleted membranes, reconstitution of phospholipase D activity required both RhoA and the 50-kDa factor. Thus, RhoA along with a non-Rho, non-ADP-ribosylation factor 50-kDa cytosolic factor are both required to reconstitute GTPγS-dependent phospholipase D activity by neutrophil plasma membranes.

Reconstitution and characterization of the human neutrophil respiratory burst oxidase using recombinant p47-phox, p67-phox and plasma membrane.

Human neutrophil respiratory burst oxidase (NADPH-oxidase) activity can be reconstituted in a cell-free system consisting of plasma membrane, cytosol and an anionic amphiphile [e.g., sodium dodecyl sulfate (SDS) or arachidonate]. Herein, we report reconstitution of oxidase activity using isolated neutrophil plasma membrane together with purified recombinant p47-phox and p67-phox which had been produced using a baculovirus expression system. Activity required an anionic amphiphile (SDS or arachidonate) and was potentiated by diacylglycerol and GTP gamma S. Serial washes of the plasma membrane failed to affect its ability to reconstitute activity, indicating that a dissociable membrane component was not present. The Km for NADPH, 43 microM, was the same as that determined using cytosol in place of recombinant factors. The EC50 values for p47-phox and p67-phox under optimal activation conditions were 220 nM and 80 nM, respectively, indicating a relatively high affinity of these components in an activation complex. Since neither cytosolic component contains a nucleotide binding consensus sequence, these data indicate that the NADPH binding component of the oxidase resides in the plasma membrane.

Expression of p47-phox and p67-phox proteins in murine bone marrow-derived macrophages: Enhancement by lipopolysaccharide and tumor necrosis factor α but not colony stimulating factor 1

We have investigated the relationship between the expression of the p47‐phox and p67‐phox cytosolic components of the NADPH oxidase and priming of the macrophage respiratory burst. Western blot analysis revealed that murine bone marrow‐derived macrophages (BMM) contain immunoreactive proteins detected by antisera raised against recombinant human p47‐phox and p67‐phox. Priming BMM by exposure to tumor necrosis factor or (TNF‐α) or lipopolysaccharide (LPS) increased the levels of p47‐phox and p67‐phox. Colony‐stimulating factor 1 (CSF‐1), which we previously found to have a negative effect on the priming of murine macrophages, had no effect on the level of p47‐phox but down‐regulated that of p67‐phox. Our results suggest that the regulatory effects of LPS, TNF‐α, and CSF‐1 on the respiratory burst of BMM may be due to modulation of the expression of the p47‐phox and p67‐phox cytosolic components of the NADPH oxidase. J. Leukoc. Biol. 55: 530–535; 1994.

A carboxy-terminal peptide from p47-phox is a substrate for phosphorylation by protein kinase C and by a neutrophil protein kinase

Agonist-activated phosphorylation of neutrophil proteins including p47-phox, a cytosolic component of the respiratory burst oxidase, has been implicated in the signal transduction cascade which leads to activation of the superoxide generating respiratory burst. We have previously reported (J. Biol. Chem. 265, 17550-59) that in a cell-free activation system consisting of cytosol plus plasma membrane from human neutrophils, diacylglycerol acts synergistically with an anionic amphiphile such as sodium dodecyl sulfate (SDS) to augment superoxide generation and assembly of the oxidase, and that p47 phosphorylation can occur under these conditions. Herein, we show that a peptide corresponding to a carboxy terminal sequence of p47-phox is a substrate for phosphorylation both by purified protein kinase C (a mixture of alpha, beta, and gamma forms) and by a distinct kinase or kinases present in neutrophil cytosol. Based on its activator requirements, the neutrophil kinase differs from classical protein kinase C, but may be a protein kinase C variant, based on inhibition by a protein kinase C peptide. Although in the cell-free system phosphorylation occurs under conditions which are similar to those for activation of superoxide generation, phosphorylation is not required for activation (1). Rather, protein assembly or aggregation which occurs under activation conditions may also promote phosphorylation.

Functional differences in human neutrophils isolated pre‐ and post‐prandially

Activated polymorphonuclear leukocytes have been associated with neoplasia, atherogenesis and reperfusion injury. Since some of these conditions are also correlated with dietary fat, we examined the functional characteristics of leukocytes isolated from subjects before and after consumption of a lipid‐rich meal. There was up to 2‐fold greater superoxide generation in response to agonists in leukocytes obtained post‐prandially; the maximum increase was observed about 4 h after eating and followed the peak (2–4 h) in serum triglycerides. Neutrophils isolated post‐prandially also exhibited impaired chemotaxis and defective bacterial killing, but normal phagocytosis. These findings provide a new variable that should be considered in studies of leukocytes.

Stabilization of the superoxide-generating respiratory burst oxidase of human neutrophil plasma membrane by crosslinking with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide

The superoxide-generating respiratory burst oxidase (NADPH-oxidase) of neutrophil plasma membranes is known to be highly unstable. In an attempt to stabilize the enzyme, we investigated the effect of crosslinking with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The stability of superoxide-generating activity of plasma membrane was significantly enhanced by crosslinking. The half-life (t1/2) of the activity at 37 degrees C in the absence of crosslinker was about 2 min. Crosslinking extended the t1/2 significantly. Crosslinked material exhibited a biphasic loss of activity: about half was lost in each phase with respective t1/2 values of 20 and 240 min. The lifetime of the crosslinked material at 37 degrees C was further extended (about sixfold) with 30% glycerol, and the crosslinked material was completely stable for more than 2 weeks if stored on ice. Crosslinking also stabilized the activity to the effects of high salt and detergent, both of which have inactivating effects on the oxidase. In addition, crosslinking stabilizes not only the Vm but also the Km of the enzyme, which was noted to increase upon storage in the absence of crosslinking. Unlike the native material, the crosslinked oxidase failed to be stimulated (and in fact was inhibited) by phosphatidylserine, recently reported to be an activator of the oxidase (Tamura et al. (1988) J. Biol. Chem. 263, 17,621-17,626). The crosslinked plasma membrane provides a useful stabilized system for kinetic studies. When the activated plasma membrane was treated with EDC, the stabilized oxidase could not be solubilized effectively using detergents, since greater than 95% of the activity remained with the pellet following centrifugation, perhaps due to crosslinking to the cytoskeleton. However, when the activity was first detergent-solubilized, the soluble activity was also stabilized by EDC. This solubilized, crosslinked material may provide useful starting material for subsequent isolation and characterization of a stabilized active NADPH-oxidase.

Enhancement of phospholipase D activity by overexpression of amyloid precursor protein in P19 mouse embryonic carcinoma cells

It has been shown that phospholipase D (PLD) activity is stimulated by the beta-amyloid protein in neuronal cells. The aim of this study was to determine whether overexpression of the amyloid precursor protein (APP) affects the activity and the level of PLD expression in P19 embryonic carcinoma cells. We observed that the unstimulated basal PLD activity was higher in wild-type APP(695)-transfected cells than in non-transfected control cells. The protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), has been shown to activate PLD. PMA-stimulated PLD activity was 3-fold higher in the APP overexpressing cells than in the control cells. P19 cells express two distinct PLD isozymes, PLD1 and PLD2. The level of PLD2 expression was increased by APP overexpression. Although the PKC inhibitor, GF109203X, inhibited PMA-stimulated PLD activity, it did not affect the high basal PLD activity induced by APP overexpression. Neuronal differentiation of the P19 cells by retinoic acid did not affect the basal or PMA stimulated-PLD activity. Interestingly, APP overexpression in the differentiated P19 cells also led to an increase in PLD activity. The PLD activity of the P19 cells is apparently regulated by amyloid protein through both PKC-dependent and -independent mechanisms.

Expression of recombinant myeloperoxidase using a baculovirus expression system

Myeloperoxidase (MPO) is a glycosylated heme-containing enzyme present in the azurophilic granules of normal human polymorphonuclear neutrophils. This enzyme plays a major role in the microbicidal activity of the host defense system by catalyzing the formation of the potent oxidant, hypochlorous acid. Although the amino acid sequence of MPO has been deduced from the cDNA, the structural basis for the observed heterogeneity of this enzyme is not known. Furthermore, the nature of the prosthetic group and its mode of linkage to the apoprotein has not been determined. To address questions regarding the structural features of MPO, which arise during the complex posttranslational processing of this enzyme, we utilized a baculovirus system to express MPO in Sf9 insect cells. Two glycosylated, single-chain precursor species of MPO were observed: an 84 kDa species that was secreted and a 74 kDa species that was cell-associated. This is the first report of an expression system in which a cell-associated MPO precursor undergoes posttranslational proteolytic processing.

Dexamethasone enhances phospholipase D activity in M-1 cells.

Phospholipase D (PLD) is an enzyme involved in signal transduction and widely distributed in mammalian cells. The signal transduction pathways and role for phospholipid metabolism during hormonal response in cortical collecting duct remain partly undefined. It has been reported that dexamethasone increases transepithelial transport in M-1 cells that are derived from the mouse cortical collecting duct. We investigated the expression and activity of PLD in M-1 cells. Basal PLD activity of M-1 cells cultured in the presence of dexamethasone (5 µM) was higher than in the absence of dexamethasone. Dexamethasone and ATP activated PLD in M-1 cells but phorbol ester did not stimulate PLD activity. Vasopressin, bradykinin, dibutyryl cyclic AMP, and ionomycin were ineffective in activating PLD of the cells. The PLD2 isotype was detected by immunoprecipitation but PLD1 was not detected in M-1 cells. Addition of GTPγS and ADP-ribosylation factor or phosphatidylinositiol 4,5-bisphosphate to digitonin-permeabilized cells did not augment PLD activity. In intact cells PLD activity was increased by sodium oleate but there was no significant change between dexamethasone treated- and untreated cells by oleate. These results suggest that at least two types of PLD are present in M-1 cells and PLD plays a role in the corticosteroid-mediated response of cortical collecting duct cells.

[27] Neutrophil phospholipase D: Inhibition by Rho-GDP dissociation inhibitor and stimulation by small GTPase GDP dissociation stimulator

Publisher Summary Neutrophils contain phospholipase D, which is activated by receptor-coupled agonists, such as f-Met–Leu–Phe and protein kinase C activators, such as phorbol esters. Phospholipase D catalyzes the hydrolysis of phospholipids into phosphatidic acid, and it has been implicated in signal transduction in the neutrophil as well as in a variety of other cells. Neutrophils are isolated based on the procedure by Pember et al. In this procedure, blood (450 ml) is obtained by venapuncture in a 600-mL transfer pack container containing 10 mL of 0.1 M EDTA, pH 7.4/0.9% of NaCl. Blood is then diluted with 180-mL Hespan and is allowed to settle in plastic cylinders for one hour at room temperature. At no time during the preparation are cells allowed to come into contact with glass. The chapter explains the labeling of cells and isolation of membrane and cytosolic fractions. A variety of assays are available for measuring phospholipase D activity, which include the formation of labeled phosphatidic acid, the release of a labeled choline headgroup, and transphosphatidylation.